This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pathogen defense in plants is mediated by the concerted action of multiple pathogen-induced, and constitutive pathways. These include systemic acquired resistance (SAR) that is dependent upon pathogen-triggered accumulation of endogenous salicylic acid (SA) and the downstream NIM1 (also called NPR1) gene in Arabidopsis thaliana that shows homology to the mammalian innate immunity regulator Ik-B. SAR-independent defense pathways also play key roles in plant defense. Together, these pathways constitute the plant innate immune system. Our recent research focus has been on elucidating SAR-independent branches of the plant defense network. Most of the current research in the Delaney program is oriented around two project areas. The first involves characterization of the arabidopsis F-Box protein SON1 that we discovered in a forward genetic screen as a negative regulator of a novel, SAR-independent defense mechanism, which is effective in preventing growth of a broad range of plant pathogens, including pathogenic oomycetes, bacteria and fungi. The other main project is directed toward the cloning and characterization of a second gene called NIP1 that we discovered in a genetic screen as required to full resistance to an important oomycete pathogen called Hyaloperonospora parasitica, an obligately biotrophic pathogen (i.e. cannot grow in absence of the plant) that is related to several important pathogens of crop plants and tree species (e.g. potato late blight, tobacco blue mold, sudden oak death, etc.). Unlike SON1-regulated disease resistance, NIP1-dependent defenses are specific to H. parasitica, and do not extend to other pathogens of arabidopsis. Our work with SON1 is guided by its structure, which shows it to contain an F-box motif, a conserved element found within a multitude of eukaryotic proteins that are subunits of SCF-class, E3 ubiquitin ligases, which function in the identification of and binding to cellular proteins, transfer of ubiquitin (Ub) moieties to the bound protein, and subsequent ubiquitin-mediated proteolysis of the modified protein. The F-box protein component of an SCF complex plays a recognition role, conferring specificity to Ub-mediated proteolysis, such that a specific SCF complex targets just a few proteins for destruction. This paradigm has led us to postulate that SON1 is a component of an SCF complex, and that it performs the role of recognizing cellular proteins that are targeted for Ub-modification and thus destined for proteolysis. Because mutations in the SON1 gene activate a novel disease resistance pathway, we further speculate that SON1 acts to negatively regulate (through proteolysis) a positive regulator of disease resistance. Accordingly, by this model, son1 mutant plants would accumulate this putative positive regulator of resistance, and express disease resistance. Our research in the past year has focused upon exploring this concept, through biochemical and genetic studies involving SON1 and the son1 mutant. Notably, plants contain very large families of F-box proteins (approx 650 in Arabidopsis and rice), suggesting that this mechanism for control of protein stability is widespread and elaborate in plants. Our research objectives for the SON1 project fall into four principle areas: i) elucidate son1-mediated resistance to determine if it requires already known defense pathways in addition to SAR;ii) refine the transcriptome of plants expressing son1-mediated resistance;iii) establish whether SON1 protein assembled into a canonical SCF complex (i.e. with components related to yeast SKP1, cullin, and Rbx, the subunits with an F-box protein that constitutes an SCF complex);and iv) identify a substrate protein(s) that is recognized by SON1. Our progress toward these objectives is outlined: i) We previously showed that salicylic acid mediated defense pathways were not required for son1-mediated resistance. To test whether defense pathways mediated by ethylene (ET) or jasmonic acid (JA) accumulation were required for son1-mediated resistance, we constructed multiple mutant constructs that contains the son1 mutation (that confers pathogen resistance) with mutations that affect ET (ein2) or JA (jar1) signaling, with or without the SAR-blocking mutation nim1-1. We found that the double (son1 ein2-1 and son1 jar1-1) and triple (son1 nim1-1 ein2-1 and son1 nim1-1 jar1-1) mutants exhibited equal disease resistance against H. parasitica and Pseudomonas syringae pv. tom. strain DC3000 as did the son1 or son1 nim1-1 parental lines, indicating that resistance in these plants does not require ET or JA signaling as defined by the mutations tested. To determine whether son1 and son1 nim1 plants responded normally to ET and JA, we examined the ET-induced seedling triple response and jasmonic acid inhibition of root elongation , two well established measures of plant responses to these compounds. Our assay results showed that son1 and son1 nim1 plants responded like the WT positive controls to these signaling compounds, while the included ein2-1 and jar1-1 negative controls showed the expected lack of response to ethylene or jasmonic acid, respectively. Together with our previous studies showing SA-independence, these studies indicate that son1 resistance does not involve defense pathways that are regulated by SA, JA or ET, and that son1 plants have intact (WT) responses to these plant hormones ii) To examine the transcriptome of son1 and son1 nim1 plants (both genotypes are disease resistant), we conducted DNA microarray (Affymetrix whole genome, ATH1 arrays) experiments using probes prepared from mRNAs obtained from wild type, son1 and son1 nim1 plants before and 4 days after exposure to H. parasitica. One of the initial observations was that very few genes were elevated (16 genes more than 2x) or repressed (32 genes less than 1/2-fold) in son1 nim1 (resistant) plants compared to WT, and even in son1 NIM1 (i.e. wild type for NIM1/NPR1), a small number of genes were elevated (35 more than 2x) compared to the INA induced WT that expressed over 10 times more induced genes (435 more than 2x). An important goal in this study was to identify gene expression traits that correlate with the SAR-independent disease resistance observed in son1 and in son1 nim1 plants, with the intent of later establishing the significance of that gene expression through the functional analyses of those genes. We have begun those functional analyses, by obtaining from the arabidopsis community stock centers insertional mutations in many of the genes identified. We are now combining those mutations with son1, and making both loci homozygous so we can assess whether disease resistance is compromised by the insertional mutations. Our intent is that this approach can allow us to identify genes involved in son1-mediated resistance. iii and iv) For our efforts to establish whether the SON1 protein assembles into a canonical SCF complex, and to identify a substrate for a putative SCF-SON1 complex, we used biochemical as well as genetic approaches. Our strategy is to seek to identify a plant SKP1-like protein that interacts with SON, which would provide evidence that SON1 exists within an SCF complex (arabidopsis has 20 SKP1-like proteins called ASK1-ASK20). To identify potential substrates of a putative SCF-SON1 complex, we anticipate that SON1 can be used as a 'handle'to identify a substrate protein, since the C-terminus of many F-box proteins has been shown to have this role in substrate recognition and binding. In our biochemical experiments, we expressed in E. coli, a GST-SON1 fusion protein, which was incubated overnight with leaf protein extracts under conditions favorable for recovery of interacting proteins. SDS-PAGE showed a major SON1-coprecipitating protein species that was identified by protein-MS with the assistance of Dr. Dwight Matthews and his colleagues. The recovered band was unambiguously identified as the large subunit of RuBP Carboxylase, a leaf protein involved in photosynthesis. Though we conducted measures to avoid artifacts (e.g. multiple preclearings of leaf extracts to remove GST or glutathione-S-transferase-interacting proteins), we are not confident that the recovery of RuBPCase is due to an authentic, in planta interaction with SON1, given the abundance of this protein in plants. As an alternative method to identify proteins that interact with SON1, we have used the yeast 2-hybrid (Y2H) system. A SON1 cDNA was cloned into the yeast 2-hybrid pGBKT7 bait vector (Clontech Laboratories, Inc.) to create a SON1-Gal4 DNA-binding domain fusion protein, that serves as a Y2H "bait." Screens for SON1-interacting proteins were conducted by co-transformation of yeast strain AH109 (Clontech) with pGBKT7-SON1 and 2-hybrid "prey" cDNA libraries that contain cDNAs fused to the Gal4 transcriptional activation domain (AD). We used three independent yeast cDNA libraries to construct prey libraries. This approach led to the reproducible recovery of three cDNAs that encode proteins that interact in this assay with SON1, but do not interact with various negative controls that we performed. The three SON1-interacting protein (SIP) genes include a dentin-like protein (At3g54500), a zinc-finger protein (At1g20110), and PETC (At4g03280), a nuclear gene product that functions in chloroplastic electron transport. The significance of the interaction in the Y2H of these proteins with SON1 is not yet clear, though we are pursuing these leads by performing deletion analysis of SON1 to determine which regions of the protein interact with each of the SIPs. The deletions are currently in testing, in directed Y2H assays in which a deletion version of SON1 (as the bait) is cotransformed into yeast with one of the SIP fusion genes. This has allowed us to narrow the region of interaction within SON1 to the carboxyl terminal third of the SON1 which is sufficient for its interaction with the PETC protein. This region of SON1 contains a conserved region identified in Pfam as the FBA1 domain, which has no known function. Our research may indicate that is has a role in protein-protein interaction. We are continuing this analysis with each SIP, with the aim of defining regions within SON1 that interact with potential substrate. Our biochemical and Y2H experiments have not identified an ASK protein (plant SKP1-homolog) that interacts with SON1. To more directly test for a specific interaction between SON1 and an ASK, we have cloned most of the 20 arabidopsis ask proteins into Y2H vectors, so that an ASK-Gal4 activation domain fusion protein in generated. These are being tested in the Y2H directly against the SON1 bait, to determine whether any of the known ASKs interact with SON1. At this time we do not have positive evidence for that interaction, though we have just completed the cloning of almost all members of the set of 20 ASK proteins, and will perform those assays in the next few weeks. An important question exists regarding SON1-regulated resistance, as to where in the plant, and under what circumstances, is this resistance normally expressed. WE do know that in mature green leaves, it is not expressed, in a SON1 (WT) plant. Therefore, to gain information on the transcriptional control of SON1, we generated SON1 promoter-reporter transgenic plants that will display the expression pattern of this gene, as well as SON1-GFP translational fusions that will help us examine subcellular localization of SON1. Finally, we have performed steps toward creation of a protoplast transient assay system that will allow is to study SON1 activity in vivo, by monitoring its ability to genetically complement a son1-1 mutant protoplast, as indicated by induced expression of a SON1-dependent promoter coupled to a luciferase reporter gene. We have use a similar assay in past work to examine NIM1 activity in vivo. Together this work is aimed at characterizing an SAR-independent pathogen defense pathway that contributes to plant innate immunity. In a separate project, we are positionally cloning an arabidopsis gene called NIP1 that plays a role in the SAR-independent resistance against H. parasitica. Plants carrying the nip1 mutation permit growth of an isolate of this pathogen that the plant is normally able to detect and against which mount an effective defense. Therefore, we postulate that NIP1 is involved in either pathogen detection, signal transduction after detection, or in production of an effector of resistance. We have mapped NIP1 to an approximately 17 cM region on chromosome III of Arabidopsis, using a mapping population of approximately 416 mutant plants identified from a screen of 12,000 F2 plants from a mapping cross. Of the 416 plants, 120 have been analyzed using PCR-based molecular markers that distinguish alleles (e.g. SNPs, RFLPs) between the mapping-cross parents (A. thaliana accession Ws-0 nip1 versus La-0 NIP1). This allowed linkage to be established between NIP1 and flanking markers 8 and 9 cM away. For reference, 17 cM corresponds to ca. 4.2 mbp, or 950 ORFs, based on genome-wide estimates of these values in A. thaliana;a map unit (cM) in this species corresponds to approx 250 kbp, and the gene density across the genome is a fairly consistent average of 4-5 kbp per gene, though these are average values and the range can varies widely. We are now narrowing the interval that encompasses the NIP1 gene to a region small enough to make feasible a DNA sequencing approach to evaluate candidate loci within the interval. This will require a high-resolution map of the NIP1 locus to an approx. 40 kbp region, which is likely to corresponds to about 10 ORFs. We will then conduct comparative sequence analysis of candidate genes in mutant and wild-type plants. To narrow the interval that includes NIP1, we are identifying additional molecular markers within the 17 cM interval, and mapping crossover points in this region that we have identified within our mapping population. Additional F2 plants are also being characterized to identify additional crossover plants that are useful to us. We have also taken an additional approach to identify by PCR F2 plants within our mapping population that have informative crossover events between the two markers that flank NIP1, which has led us to identify several dozen new crossover plants that we are now preparing to phenotype to determine the genotype of the NIP1 locus in these plants. This approach can provide the genetic resources we will need to narrow the minimal region that contains NIP1. Candidate gene will be tested in planta by using complementation tests of mutant plants that have introduced into them a wild-type copy of a gene believed to be responsible for the mutant phenotype. We have expertise in this approach to gene isolation, having used it to previously clone A. thaliana DET1, NIM1 and SON1 genes. Both projects have excellent potential to inform us to novel pathways that plants use to fight infection and prevent disease. The SON1 project also may provide important information to the function of a novel subfamily member of the very large (650 member) F-box protein family in plants. Knowledge of the molecular nature of the nip1 mutation and involved gene is also exciting to us, as few pathogen-specific susceptibility mutants have been isolated in plants (outside of plants with mutations in the sensory function encoded by so-called plant "Resistance" genes). Because nip1 has a H. parasitica-specific defect in resistance, it has promise to inform us to the mechanisms by which plants combat this important class of pathogens. Delaney submitted a renewal NSF proposal to support three years of additional research on SON1 and related members of the A. thaliana F-box protein family that are similar to SON1 ($577,523 requested). This SON1-containing subfamily (ca. 100 genes) is united by presence of a C-terminal "FBA1" domain of unknown function. The proposal describes work on SON1 and about ten other members of the FBA1-containing gene subfamily to examine three hypotheses: 1) that SON1 functions to activate disease resistance at a specific time in development, location within the plant, or under specific circumstances (that will be examined);2) that some members of the large FBA1-FBX family of FBX proteins have important and identifiable biological functions;and 3) the FBA1 domain plays a role in those functions, and constitutes a biologically relevant, protein-interaction motif. Publications Delaney, T.P. (2008) Plant Defense Against Pathogens. In Yearbook of Science and Technology, McGraw-Hill. pp. 289-292. St.-Pierre, B. and Delaney, T. P. (2008) Molecular and cellular characterization of the Arabidopsis SON1 F-box factor. Phytopathology 98: Supp. 195. In prep: Argueso, C.T., Olarte, R., and Delaney, T. P. The SON1-regulated defense pathway in Arabidopsis does not rely on salicylate, jasmonate or ethylene defense pathways and is associated with altered expression of genes. In prep: Argueso, C.T., Li, Z. and Delaney, T. P. The arabidopsis NIP1 gene is required for full expression of race-specific resistance against Hyaloperonospora parasitica. Mentor Summary: Cory Teuscher Previously we addressed the issues associated with submitting both competing renewal and new grant applications. A competing renewal application was submitted. Reasonable minimal expectations were again stressed: one manuscript in press, one in review, and at least one NSF/NIH grant application per year until funding is obtained. Additionally, we discussed a more focused approach to his research program, particularly with respect to productivity.